JP6198741B2 - Compounds for treating influenza - Google Patents

Description

  The present invention relates to novel neuraminidase inhibitors for treating influenza (flu) and methods for their preparation.

  Influenza is a typical respiratory viral infection. In the United States alone, it is believed that 10% to 20% of the population is infected with this virus every year. According to WHO, the virus is responsible for the disease of 3 to 5 million people, and the annual number of deaths caused directly by either this virus or secondary infection is 250,000 to 500,000. It is. Randomly occurring epidemics or epidemics have significantly increased infection levels and deaths. In the last century, three major influenza pandemics (1918-19 H1N1 virus ("Spanish influenza"), 1957 H2N2 virus, and 1968 H3N2 virus caused a cumulative total of about 50 million deaths "Spanish influenza" is the most severe pandemic representative to date, causing the death of about 20 million people in the first year alone, the last pandemic happened in 2009, Known as “influenza” (H1N1, “swine flu”), it was relatively harmless in terms of death, however, in the last few years, particularly highly pathogenic “bird flu virus” (H5N1) Especially because it is easily transmitted from animals to humans, It has become a cause just in case material.

At present, there are two main (and approved) therapeutic approaches for treating influenza or preventing infection:
(1) Vaccination (2) Use of antiviral active ingredients:
a. M2 channel blocker, adamantane derivative (amantadine, rimantadine)
b. Neuraminidase inhibitor

However, neuraminidase inhibitors have significant advantages over M2 channel blockers:
(1) Since it is effective against influenza A and B, it has a wide range of antiviral effects. Adamantane derivatives are only effective against influenza A;
(2) rarely induces strong viral resistance mechanisms;
(3) Excellent tolerability;
(4) Excellent effect of reducing respiratory events.

  Influenza viruses are composed of an outer membrane that surrounds a nucleocapsid. The glycoproteins hemagglutinin and neuraminidase are located on this outer membrane. After the virus infects cells and viral replication begins, new virus particles (so-called virions) coated with sialic acid are formed. As long as sialic acids are bound to virions, they aggregate with hemagglutinin residues of other virions. These virion aggregates can no longer invade and infect other cells. Thus, one of the functions of neuraminidase is to cleave sialic acid residues from virions so that they can circulate freely in the body and infect other cells. Thus, the use of neuraminidase inhibitors represents a therapeutic approach in the treatment of influenza.

  Meanwhile, several very potent neuraminidase inhibitors have been developed and received regulatory approval for the treatment of influenza, such as zanamivir, oseltamivir and peramivir.

In the field of neuraminidase inhibitors, many research and development projects have been conducted to identify new active ingredient candidates. In addition to the development of new lead structures, there are a number of ongoing efforts aimed at optimizing or modifying the known structures of oseltamivir and zanamivir. The general goal of all exploration is always to obtain compounds with improved efficacy or bioavailability. By doing so, it is impossible to develop a compound that fully satisfies both criteria. However, various modifications to oseltamivir and zanamivir have been described that result in highly effective compounds against influenza. The following is a summary of the modifications already described:

1st place:
Many studies have referred to modification of the isopentyl side chain. A number of modifications are now known to help reduce or increase lipophilicity through side chain modifications, and these have only a minor effect on the affinity for neuraminidase. ^In addition, it has been shown that hydroxylation can be performed at each position of the isopentyl side chain. ^{Based on two} structurally similar zanamivirs, various substituted aliphatic radicals and various (hydroxylated and non-hydroxylated) cyclic side chains have been shown to be acceptable at this position. ³ Li et al. Extended this approach by rational design of 46 oseltamivir analogs, and showed that substitution at position 1 was meaningful in increasing affinity for neuraminidase. The study was also able to identify a variety of substituents-both aromatic and aliphatic. Furthermore, oxygen could be replaced with nitrogen. ⁴ These data demonstrate that the structural requirements at this position of the molecule are relatively non-specific and that various structures are acceptable. Accordingly, many other active derivatives of oseltamivir can be synthesized by modifying the isopentyl functional group and can be obtained according to the described procedure of the present invention below.

4th place:
For example, modification of position 4 of oseltamivir was evaluated by Wang et al. It has been shown that substitution of this position is possible to optimize ⁶ neuraminidase affinity. These substituents were linked to the cyclohexene skeleton via carbon, oxygen or nitrogen, respectively.

5th place:
Various modifications are also possible at this position. Thus, for example, it has been shown that the carboxyl functionality can be replaced with other similar functionality (phosphonic acid, phosphonic acid ester, phosphonic acid amide, etc.) without losing its effectiveness. ^It can also be envisaged to replace the ^7,8 carboxyl carbon with sulfur to the corresponding sulfonic acid derivative.

6th position and skeleton:
Further modifications relate to the cyclohexene skeleton of oseltamivir. Various research groups were interested in replacing this central structural feature with other structures without changing the ring structure of the element. It has been found that aromatic and 5-membered ring systems can have similar effectiveness. ^{9,10 In} addition, various aliphatic substituents were tested at the 6-position, indicating their affinity for neuraminidase. It can also be envisaged to use heteroatoms in this ring system, as was done with zanamivir. Thus, skeletal modification demonstrates not only that the size of the central structural element can be changed, but also that the double bond of oseltamivir cyclohexene is not essential. Validating both the number and position of double bonds results in potent neuraminidase inhibitors. ^{10, 11}

  A great deal of development effort in the field of neuraminidase inhibitors has identified a number of oseltamivir-like structures that have similar effects to oseltamivir. However, none of these derivatives are superior to oseltamivir in terms of their overall properties, and none of these analogs are approved as active ingredients.

  Critical to the success of a therapeutic agent is its pharmacokinetic profile, which is affected by solubility, the ability to activate potential prodrugs to their active form, and the formation of protein bonds. Its stability under physiological conditions is equally important.

  The oral bioavailability of a therapeutic agent, drug or drug, which indicates how much the active ingredient is absorbed after oral administration, is also critical. Oral administration is one of the easiest to use compared to other dosage forms. For example, sterilization required at the time of infusion or injection is not necessary. In addition, dosage and application are much easier than in the inhaled pharmaceutical form.

  In Germany, zanamivir and oseltamivir are currently approved for the treatment of influenza. There is already an available oral dosage form of oseltamivir (Tamiflu®), but the use of zanamivir (Relenza®) is only 2% inhalation because the bioavailability after oral administration is only 2% Is possible. In addition, in other countries, peramivir is approved as an emergency medication for intravenous administration in pandemic events. Recently, in Japan, laninamivir (Inavir®) was approved for inhalation therapy of influenza.

  In the case of a pandemic event, oral dosage forms may have advantages over inhaled or intravenous dosage forms.

  A significant problem with anti-influenza drugs is that influenza viruses, particularly the major viral antigens (hemagglutinin and neuraminidase), are rapidly mutated to rapidly develop resistance to existing drugs. This mutation is particularly prominent in influenza A virus. Sudden so-called “antigen discontinuity mutations” can occur where it is necessary to adjust the vaccine against influenza annually (seasonally). In particular, in recent years, the cause of concern is the increasing incidence of oseltamivir resistance A (H1N1) having the H274Y mutation. Furthermore, further new resistance mechanisms have been discovered following several known mutations.

  Oseltamivir (Tamiflu®) should be ineffective against further viral resistance and the effective amount of orally administered substance effective against influenza is unknown.

  The object of the present invention is therefore to find neuraminidase inhibitors which are also effective against oseltamivir resistant virus strains starting from the structure of oseltamivir.

  Furthermore, it is an object of the present invention to provide neuraminidase inhibitors that exhibit an improved pharmacokinetic profile.

  Another object of the present invention is to provide a neuraminidase inhibitor having excellent bioavailability.

  These objects are achieved by the embodiments characterized in the claims and herein. The subclaims and examples provide advantageous details of the invention.

（式中、Ｒ^{１，４，５}は、同一でも異なっていてもよく、−Ｈ、１〜１２の鎖長を有する分岐状または非分岐状、飽和または不飽和、置換または非置換炭化水素鎖であり、Ｒ^２はＨであるか、または例えばＷａｎｇ ｅｔ ａｌ．^６もしくはＰａｒｋ ａｎｄ Ｊｏ（Ｐａｒｋ ａｎｄ Ｊｏ，Ｅｕｒ．Ｊ．Ｍｅｄ．Ｃｈｅｍ．４５（２０１０），５３６−５４１）（これらの開示は、前述の説明での使用によって本明細書に組み込まれている）に記載されているような置換基、特に、

ＣＨ_２ＯＨ、ＣＨ_２ＮＨ_２、ＣＨ_２ＣＯＮＨ_２、ＣＨ_２ＣＨ_２ＣＯＮＨ_２、１〜１２の鎖長を有する分岐状または非分岐状、飽和または不飽和、置換または非置換炭化水素鎖からなる群より選択される置換基であり、
Ｒ^３は、Ｈ、ＯＨ、ＯＲ^１、ＯＣＯＯＲ^１またはＣＯＯＲ^１であり、
Ｒ^６は、Ｈ、Ｒ^１、ＮＨ_２、ＮＨＲ^１、Ｎ（Ｒ^１）_２、ＮＨＣＯＯＲ^１であり、
Ｘは、ＯＲ^１、ＳＲ^１、ＮＨＲ^１またはＮ（Ｒ^１）_２であり、
Ｚは、Ｃ、Ｓ、ＳＯ、Ｐ、ＰＯを表し、
ここで、Ｒ^１の任意の置換基は、フッ素、塩素、臭素およびヨウ素、酸素、硫黄、アルコキシ、アシルオキシ、ヒドロキシル、メルカプト、シアノ、ニトロおよびチオアルコキシ基からなる群より選択されるか、または保護基でブロックされた官能基である）の、インフルエンザ感染症を処置するためのインフルエンザノイラミニダーゼ阻害剤、およびその薬学的に許容し得る塩、溶媒和物、Ｒ／Ｓエナンチオマー、またはプロドラッグによって達成される。 In some embodiments, the object of the present invention is a general structural formula

Wherein R ¹ , ^{4 and 5} may be the same or different and are —H, branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12 R ² is H or, for example, Wang et al. ⁶ or Park and Jo (Park and Jo, Eur. J. Med. Chem. 45 (2010), 536-541) (the disclosures of these are: Substituents such as those described in the above description, which are incorporated herein by reference,

CH ₂ OH, CH ₂ NH ₂ , CH ₂ CONH ₂ , CH ₂ CH ₂ CONH ₂ , consisting of branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1-12 A substituent selected from the group;
R ³ is H, OH, OR ¹ , OCOOR ¹ or COOR ¹ ;
R ⁶ is H, R ¹ , NH ₂ , NHR ¹ , N (R ¹ ) ₂ , NHCOOR ¹ ;
X is OR ¹ , SR ¹ , NHR ¹ or N (R ¹ ) ₂ ;
Z represents C, S, SO, P, PO,
Here, the optional substituent of R ¹ is selected from the group consisting of fluorine, chlorine, bromine and iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro and thioalkoxy groups, or protected Functional group blocked with an influenza neuraminidase inhibitor for treating influenza infections, and pharmaceutically acceptable salts, solvates, R / S enantiomers, or prodrugs thereof. The

  In a preferred embodiment, the present invention relates to amidoxime (3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidamide] -3- (1-ethylpropoxy) cyclohex-1- En-1-carboxylic acid ethyl ester or hydroxyguanidine (3R, 4R, 5S) -4-acetamido-5- [N- (N′hydroxy) -guanidino] -3- (1-ethylpropoxy) cyclohex-1- The present invention relates to influenza neuraminidase inhibitor compounds of the present invention for treating influenza infections, which are en-1-carboxylic acid ethyl esters, and pharmaceutically acceptable salts, solvates or prodrugs thereof.

Based on the basic structure of oseltamivir, the compounds of the present invention provide new influenza neuraminidase inhibitors.

のオセルタミビルまたはその誘導体を反応させることを含む、上記本発明の化合物を調製するための方法であって、以下の工程：
有機溶媒中、室温で、オセルタミビルをアシルヒドロキシモイルクロリドと反応させることを少なくとも含む方法に関する。 In a further aspect, the invention provides a general structural formula

A process for preparing a compound of the invention as described above, comprising reacting the oseltamivir or derivative thereof of
It relates to a process comprising at least reacting oseltamivir with acylhydroxymoyl chloride in an organic solvent at room temperature.

A method in which the organic solvent is dichloromethane is preferred. In a particularly preferred embodiment, the method of the present invention relates to an amidoxime compound (R ³ = OH) of the above general structural formula.

As an optional step, treatment of the ethyl ester functional group (R ⁴ = CH ₂ CH ₃ , Z = C) in the carboxyl functional group (R ⁴ = H, Z = C) by treatment with an alcoholic hydroxide solution. Relocation can be performed.

  Amidinium salts are preferred as amidating reagents. Preferably, S-naphthyl-methyl-aceto-amidinium-bromide was used.

  As the alcohol, an organic compound having a saturated or unsaturated, branched or unbranched carbon chain having a length of C1 to C6 is suitable, and an alcohol having a saturated branched or unbranched carbon chain having a length of C1 to C6 is used. Particularly preferred. Most preferably, alcohols of methanol, ethanol, propanol, butanol, iso-propanol and tert-butanol are used. For the purposes of the present invention, an alcoholic solution is meant herein to include one or more alcohols as defined above.

Preparation of the amidoxime (R ³ = OH) of the present invention is according to the present invention by the following steps: reacting oseltamivir with hydroxymoyl chloride (eg acetylhydroxymoyl chloride) in an organic solvent (eg dichloromethane) at room temperature. Is performed by a method including at least.

  Organic solvents are known to those skilled in the art and include, for example, chloroform, acetone, acetonitrile, methanol and the like in addition to dichloromethane.

  The present invention also comprises reacting oseltamivir (3R, 4R, 5S) -4-acetamido-5-amino-3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester or a derivative thereof. Comprising the steps of: (i) reacting oseltamivir with cyanobromide at room temperature in an organic solvent at room temperature (ii) formed with i at room temperature in dioxane To a process comprising at least one of reacting the treated cyanamide with hydroxylamine.

Specifically, the present invention relates to the above process wherein the compound is hydroxyguanidine (R ⁶ = NH ₂ , NHR ¹ or N (R ¹ ) ₂ and R ³ = OH) of the above general structural formula.

  As already explained above, the modifications made according to the present invention as shown on the basis of oseltamivir, and as a result, the methods of the present invention include, for example, oseltamivir derivatives modified at the above residues, for example, It can also be applied to oseltamivir derivatives already described in the current state of the art, so that those skilled in the art will recognize that these are equivalent embodiments of the invention shown in the examples. It can be clearly recognized.

Stability of some compounds of the invention at different pH values in mouse or human plasma. Stability of active form 3 (A)) and active form 9 (B)) in different media and at different temperatures. Plasma levels of derivative 3 after intravenous administration of derivative 3 to a total of 5 rats. Plasma levels of derivative 3 after oral administration of derivative 4. Plasma levels of derivative 3 and metabolite 11 after oral administration of derivative 4. Plasma levels of derivative 3 after oral administration of derivative 3. Plasma level of derivative 3 after oral administration of derivative 2. Plasma levels of derivative 3 after administration of an amidine neuraminidase inhibitor. Plasma levels of derivative 9 after administration of derivative 9 intravenously (iv). Plasma levels of derivative 9 after oral administration of derivative 9. Plasma levels of derivative 9 after oral administration of derivative 6. Plasma levels of derivative 9 and metabolite 12 after oral administration of derivative 6. Plasma levels of derivative 9 after application of a guanidine neuraminidase inhibitor.

In pursuit of various synthetic strategies to synthesize the neuraminidase inhibitors of the present invention, various oseltamivir derivatives with very different substitutions were simultaneously revealed. Examples of various synthetic routes leading to various active ingredients of the present invention are shown in the following summary.

  Overview of synthesis of neuraminidase inhibitors. A) Synthetic route of amidine-based oseltamivir derivative 3 and corresponding amidoxime 4; B) Synthetic route to hydroxyguanidine-based oseltamivir derivative 6; C) Additional synthetic route to hydroxyguanidine-based oseltamivir derivative (8); D) Oseltamivir- Structural formula of guanidine 9 and its ester prodrug 10

  S-naphthyl-methyl-aceto-amidinium-bromide was used as an amidating reagent for the synthesis of amidine (2, 3) (see A). It is also possible to use other reagents for preparing amidines. Various amidines are available in this way by the N-substitution of oseltamivir and by selecting alternative thioamidinium salts. Other known methods for making amidine functional groups can be used here. Preferably, amidoxime 4 was synthesized by using acetohydroxymoyl chloride.

  The synthesis of guanidine-based compounds is shown in B). Two different concepts for making substituted or unsubstituted hydroxyguanidine were thereby implemented. Thus, unsubstituted hydroxyguanidine 6 is available via the synthesis of cyanamide 5. Very different O, N-substituted hydroxyguanidines (8) can be synthesized via type 7 carbamoyl-substituted thioureas (see C).

  Oseltamivir-guanidine 9 and its ester prodrug 10 are also shown. This synthesis is described by Shie et al. [Shie, J. et al. Fang, J .; Wang, S .; Tsai, K .; Shyun, Y .; Cheng, E .; Yang, A .; Hsiao, S .; Su, C .; Wong, C .; , Journal of American Chemical Society 2007, 129, 11892-93].

（式中、Ｒ^１は、Ｈ、１〜１２の鎖長を有する分岐状または非分岐状、飽和または不飽和、置換または非置換炭化水素鎖であり、Ｒ^７は、ＨまたはＯＨであり、Ｒ^８は、Ｈ、Ｒ^９、ＮＨ_２、ＮＨＲ^９、Ｎ（Ｒ^９）_２またはＮＨＣＯＯＲ^１であり、
Ｒ^９は、１個〜４個の炭素原子の鎖長を有する分岐状または非分岐状アルキルラジカルであり、
ここで、Ｒ^１およびＲ^９の可能な置換基は、フッ素、塩素、臭素およびヨウ素、酸素、硫黄、アルコキシ、アシルオキシ、ヒドロキシル、メルカプト、シアノ、ニトロおよびチオアルコキシ基からなる群より選択されるか、または保護基でブロックされた官能基である）の新たなオセルタミビル誘導体、ならびに適切な溶媒和物、塩、Ｒ／Ｓエナンチオマーおよび／またはプロドラッグを提供することに関する。 In a further aspect, the invention provides a general structural formula:

Wherein R ¹ is H, a branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1 to 12, R ⁷ is H or OH; R ⁸ is H, R ⁹ , NH ₂ , NHR ⁹ , N (R ⁹ ) ₂ or NHCOOR ¹ ;
R ⁹ is a branched or unbranched alkyl radical having a chain length of 1 to 4 carbon atoms,
Where possible substituents for R ¹ and R ⁹ are selected from the group consisting of fluorine, chlorine, bromine and iodine, oxygen, sulfur, alkoxy, acyloxy, hydroxyl, mercapto, cyano, nitro and thioalkoxy groups Or a functional group blocked with a protecting group) and the provision of suitable solvates, salts, R / S enantiomers and / or prodrugs.

In one embodiment, the present invention relates to compounds GS4116 and GS4109, described at page 648 of a publication by Li et al. (Antimicrobial Agents and Chemotherapy 42 (1998), 647-653), wherein R ¹ is H or CH ₂ CH ₃ , R ⁷ is H and R ⁸ is NH ₂ ).

In a further embodiment, the invention relates to compounds wherein R ⁷ is OH when R ⁸ is NH ₂ .

In a further embodiment, the invention relates to compounds wherein R ¹ is not H or CH ₂ CH ₃ when R ⁷ is H and R ⁸ is NH ₂ .

  In a preferred embodiment, the present invention relates to the compound amidoxime (3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidamide] -3- (1-ethylpropoxy) cyclohexa- 1-ene-1-carboxylic acid ethyl ester or hydroxyguanidine (3R, 4R, 5S) -4-acetamido-5- [N- (N′hydroxy) -guanidino] -3- (1-ethylpropoxy) cyclohexa- 1-ene-1-carboxylic acid ethyl ester and / or pharmaceutically acceptable salts, solvates, R / S enantiomers, and / or prodrugs thereof.

In a preferred embodiment, the present invention relates to the above compounds and neuraminidase inhibitors for use as a medicament, preferably for treating influenza infection, and in one embodiment, the medicament of the present invention is a publication by Li et al. (Antimicrobial Agents and Chemotherapy 42 (1998), 647-653), compounds GS4116 and GS4109 of the above general structural formula described on page 648 (wherein R ¹ is H or CH _2). CH ₃ , R ⁷ is H, and R ⁸ is NH ₂ ). Contrary to the description of Li et al., The guanidine derivatives (3 and 9) of the present invention show very good efficacy against influenza.

  Experiments conducted in accordance with the present invention have shown that existing influenza resistance mechanisms can be defeated by exchanging amine functional groups with amidine or guanidine groups. Test data obtained in experiments conducted in accordance with the present invention from various antiviral assays show that amidine and guanidine derivatives are as potent as oseltamivir and zanamivir against influenza A (H1N1) strains Is effective against oseltamivir resistant influenza A (H1N1) strains. They are also effective against various influenza A (H3N2) strains. In addition, the compounds of the present invention exhibit surprisingly low toxicity in cytotoxicity assays, excellent solubility, excellent activation in the active form, and very good stability under physiological conditions.

  Table 1 shows the effectiveness of the substances of the invention (3 and 9) in a neuraminidase (NA) inhibition assay compared to zanamivir and oseltamivir. A detailed description of the test can be found in the description of the method.

表１の値は、３および９の両方が、ナノモル濃度の範囲で、試験したインフルエンザＡウイルス８種のＮＡ活性を阻害することを示している。ＮＡ阻害アッセイで決定したＩＣ５０値は、ザナミビルおよびオセルタミビルのものとほぼ同程度であった。

The values in Table 1 indicate that both 3 and 9 inhibit NA activity of 8 influenza A viruses tested in the nanomolar range. The IC50 values determined by the NA inhibition assay were about the same as those of zanamivir and oseltamivir.

  In contrast to oseltamivir, 3 and 9 are also effective against oseltamivir resistant isolate A / 342/2009. The 50% inhibitory concentration defined for this virus was 10 times (9) or 50 times (3) higher compared to zanamivir.

  Antiviral effects are also evident in virus production inhibition assays. The test results are shown in Table 2. Amidine active form (3) and guanidine active form (9) reduced the titer of influenza virus A / Jena / 5258/2009 by 90% and 95% in the nanomolar range. A detailed description of the test conditions can be found in the description of the method.

  There are also indications of the antiviral activity of the compounds of the invention in the cpE inhibition assay.

  The replication of the virus used in the test results in complete destruction of the host cell due to a very pronounced cytopathic effect (cpE). By adding an antiviral active substance (100 μl / well; 3 parallel / concentration, dilution factor 2), virus-induced cpE can be selectively inhibited. In the test, the treated and untreated closed cell layers were inoculated with a fixed dose of virus, and the untreated virus control resulted in complete cpE 48 hours after infection.

  Amidine active type 3 and guanidine active type 9 inhibit cpE of influenza viruses A / Jena / 5258/2009 and A / 342/2009 in a non-cytotoxic concentration range, with a 50% inhibitory concentration of 2.0 μM and 12.2 μM (derivative 3) and 1.1 μM and 4.1 μM (derivative 9). A detailed description of the test conditions for the test can be found in the description of the method.

Another advantage of the compounds of the present invention is their low toxicity. In this regard, in a cytotoxicity assay for determining the 50% cytotoxic dose (CC ₅₀ ) of a test substance in an MDCK (Madin-Derby Canine Kidney) cell layer, the amidine (3) and the guanidine active form (9) are It was found to be non-toxic in the test concentration range of 6.25 to 200 μg / ml. A detailed description of the test conditions can be found in the description of the method.

  Another preferred feature of the compounds of the invention is their excellent stability, especially under physiological conditions. This test showed that each compound was very stable within the pH range 2-9 (FIG. 1). For any of the compounds, no degradation of the compound was observed within the 6 hour test period.

Further stability studies for active forms 3 and 9 were conducted for 14 days and both compounds were shown to be stable over the test period at both 4 ° C. and room temperature (RT) under physiological pH. (FIG. 2). Storage stability in solution was measured at a concentration of 0.2 mg / ml. For this purpose, the compound is dissolved in 50 mM KH ₂ PO ₄ buffer (pH 7, ₄ ) or Aqua bidest and is at room temperature (RT) (pH 7.4) or in a refrigerator at 4 ° C. (pH 7.4 or Aqua). bidest) and stored over the test period. The concentration of the active form was measured by HPLC after 12 hours, 1 day, 2 days, 4 days, 7 days and 14 days.

  Other studies with mouse and human plasma have shown that the ester function (in all prodrugs, the ethyl ester of the carboxy function) is rapidly cleaved by plasma enzymes. Ester cleavage is an important step in in vivo activation and could be demonstrated by these incubations. Active forms 3 and 9 are not metabolized by plasma enzymes.

  A particular advantage of the compounds of the invention is their excellent solubility. Most of the compounds (2, 3, 6 and 10) are soluble at concentrations> 50 mM at all pH values studied (Table 4). In addition, all substances were found to be soluble above 50 mM at a pH value of 2. Thus, all compounds are believed to dissolve very well in the acidic environment in the stomach. In addition, however, less soluble substances (4, 9) are soluble above 20 mM at all pH values. Thus, all the compounds exhibit very good solubility properties, which is a preferred factor for using them as active ingredients. Thus, for example, in addition to oral dosage forms, there may be liquid dosage forms (injection, infusion) required in emergency medicine.

  Another advantage of the compounds of the present invention is the fact that the compounds only form very gentle plasma protein bonds. Experiments performed showed that all test compounds had less than 40% protein binding (Table 5). The clinically relevant interaction due to the newly developed prodrug and active protein binding is not expected since the risk of interaction of clinically relevant drugs increases only when the protein binding is greater than 90% .

In addition, protein binding of both active oseltamivir-amidine (3) and oseltamivir-guanidine (9) was examined in human plasma. For this purpose, human plasma was used instead of 4% albumin solution. Protein binding measured was 3.7 ± 1.4% for Compound 3 and 8.6 ± 3.0% for Compound 9. As expected, these values are somewhat higher than those obtained with the 4% albumin solution and are due to the presence of other plasma proteins (eg, α ₁ acidic glycoprotein) in addition to albumin.

  Another particular advantage of the compounds of the invention is the fact that already established prodrug concepts can be used. As esters, carboxylic acids are used in conventional form. The currently well established N-hydroxy concept was also used for the amidine and guanidine functional groups. The described prodrug forms are also interesting from the standpoint of sustained release of the active form after oral or parenteral administration.

  Prodrugs were tested in vitro for stability, solubility and activation to the active form. These results indicated that the compounds are sufficiently stable, have very good solubility, and that activation to the active form occurs to a good extent in various enzyme preparations.

  Activation of prodrugs to their active form was measured in vitro by intracellular enzyme preparations. As enzyme preparations, 9000 g supernatant, microsomes and mitochondria from human and porcine liver tissue were used. The incubation mixture consisted of 500 μM prodrug, 500 μM NADH, 1 U esterase and 0.3 mg enzyme preparation dissolved in 150 μl 100 mM phosphate buffer (pH 6.3). Incubation was for 30 minutes at 37 ° C. in a shaking water bath. Incubation was terminated by adding 150 μl of acetonitrile. Subsequently, the sample was shaken for 10 minutes and the precipitated protein was centrifuged at 10,000 rpm for 15 minutes. The supernatant was measured by HPLC.

  Table 6 shows the obtained conversion speed.

  Activation studies conducted in vitro showed that all the prodrugs developed were converted to active forms 3 and 9 (Table 4). That is, as shown in stability studies in human and mouse plasma, ester cleavage occurred, and in addition, in these incubations, reduction of amidoxime or N-OH-guanidine could be detected. In summary, it can be said that compounds 2, 4, 6 and 10 are suitable prodrugs of active forms 3 and 9. This study provides only a general proof that in vivo activation of the compound occurs. The conversion rate should be significantly higher in vivo.

  For prodrugs 2 and 10, the enzymatic hydrolysis of carboxylic acid ethyl ester was analyzed in more detail. Nonspecific carboxylesterase from pig liver was used as the enzyme source. The incubation mixture contained 200 μM prodrug and 3U esterase dissolved in 200 μl of 100 mM phosphate buffer (pH 7.4). Incubation was for 60 minutes at 37 ° C. Samples were analyzed every 15 minutes by HPLC.

  Incubation showed that both prodrugs were activated by esterase to their respective active forms. The conversion rates were 0.83 ± 0.14 nmol / min / mg protein (prodrug 2) and 1.35 ± 0.15 nmol / min / mg protein (prodrug 10).

  A particular advantage of the compounds of the present invention is their excellent bioavailability.

In a rat animal study, newly developed neuraminidase inhibitors were tested for their oral bioavailability. This indicates that all test compounds are absorbed from the gastrointestinal tract and metabolized to their active form. For clarity, the metabolism of neuraminidase inhibitors 4 and 6 is shown.

Bioavailability of oseltamivir amidoxime derivatives (2, 3 and 4) The highest plasma levels were obtained after administration of oseltamivir amidoxime derivative 4. Following oral administration, plasma levels of active form 3 in the μmolar range were measured over a 6 hour test period. In addition, the intermediate metabolite 11 metabolized to the active form 3 could be detected after administration of the derivative 4 at a low scale. The bioavailability of amidoxime derivative 4 was determined to be 31.3%. The plasma half-life of 3 is about 112 minutes (Table 2). The only neuraminidase inhibitor with oral bioavailability currently approved is oseltamivir (Tamiflu®). Animal studies of rats have shown that this compound has an oral bioavailability of 36% and a plasma half-life of 44 minutes. [E. J. et al. Eisenberg, A .; Bidgood, K.M. C. Cundy, Antimicrob Agents Chemother 1997, 41 (9), 1949-1952].

  Therefore, the newly developed compound 4 of the present invention is comparable in terms of bioavailability. The decisive advantage of this compound is, on the one hand, that it has an extended half-life (which allows long-acting plasma levels) and on the other hand its efficacy against oseltamivir-resistant influenza strains is evident It is excellent in.

  Similarly, administration of the other compounds (2, 3) showed detectable plasma levels of derivatives 3, however, these were lower than after administration of 4.

  The results of intravenous administration of derivative 3 are shown in the following table and FIG.

Average of total plasma levels after intravenous administration of derivative 3

  Oral administration of derivative 4 resulted in the following plasma levels of derivative 3.

Average of total plasma levels of derivative 3 after oral administration of derivative 4

  FIG. 4 shows the results graphically.

  After oral administration of derivative 4, derivative 11 is detected as a metabolite. This can also be inferred from FIG.

Average of total plasma levels of intermediate metabolite 11 after oral administration of derivative 4

  Oral administration of derivative 3 results in the following plasma levels: See also FIG.

Average of total plasma levels of derivative 3 after oral administration of derivative 3

  Oral administration of derivative 2 results in the following plasma levels: See also FIG.

Average of total plasma levels of derivative 3 after oral administration of derivative 2

  FIG. 8 and the following table outline the results from administration of amidine neuraminidase inhibitors (2, 3 and 4).

ＡＵＣ＝曲線下面積（ａｒｅａ ｕｎｄｅｒ ｔｈｅ ｃｕｒｖｅ）；ｔ_ｍａｘ＝最大血漿レベルが測定された時間。
ｃ_ｍａｘ＝決定された最大血漿濃度；ＭＲＴ＝平均滞留時間（Ｍｅａｎ Ｒｅｓｉｄｅｎｃｅ Ｔｉｍｅ）
ｔ_１／２＝血漿半減期；血漿半減期ｔ_１／２と同様に、ＭＲＴ（平均滞留時間：Ｍｅａｎ Ｒｅｓｉｄｅｎｃｅ Ｔｉｍｅ）は、体内における物質の滞留時間の尺度である。それは古典的な薬物動態の値であり、ＡＵＭＣ（１次モーメント曲線下面積：ａｒｅａ ｕｎｄｅｒ ｔｈｅ ｆｉｒｓｔ ｍｏｍｅｎｔ ｃｕｒｖｅ）をＡＵＣ（曲線下面積：ａｒｅａ ｕｎｄｅｒ ｔｈｅ ｃｕｒｖｅ）で割ることによって得られる。

AUC = area under the curve; t _max = time at which the maximum plasma level was measured.
c _max = determined maximum plasma concentration; MRT = mean residence time
t _1/2 = plasma half-life; as with plasma half-life t _1/2 , MRT (Mean Residence Time) is a measure of the residence time of a substance in the body. It is a classic pharmacokinetic value, obtained by dividing AUMC (area under the first moment curve) by AUC (area under the curve).

Bioavailability of oseltamivir hydroxyguanidine derivative (6, 9, 10) Analysis of plasma samples provided detectable plasma levels of derivative 9 over 6 hours after administration of all test derivatives. However, compared to oseltamivir amidoxime derivatives (see a), the plasma levels measured were surprisingly low. When the plasma concentration of derivative 9 was measured within the three-digit nanomolar range, it was about 10 times lower than that of derivative 4. After administration of derivative 6, intermediate metabolite 12 metabolized to active form 9 could be detected.

  The bioavailability of hydroxyguanidine derivative 6 was determined to be 1.7%. The plasma half-life of 9 is about 98 minutes. The oral bioavailability of the other tested guanidine-based oseltamivir derivatives (9, 10) is not significantly different from derivative 6.

  The results of intravenous administration of derivative 9 are shown in the following table and FIG.

Average of total plasma levels after intravenous administration of derivative 9

  Oral administration of derivative 9 results in the following plasma levels: See also FIG.

Average of total plasma levels of derivative 9 after oral administration of derivative 9

  Oral administration of derivative 6 results in the following plasma levels: See also FIG.

Average of total plasma levels of derivative 9 after oral administration of derivative 6

  After oral administration of derivative 6, derivative 12 is detected as a metabolite. This can also be inferred from FIG.

Average of total plasma levels of intermediate metabolite 12 after oral administration of derivative 6

  FIG. 13 shows a summary of the results of administration of a guanidine neuraminidase inhibitor (6, 9, 10).

Materials and Methods: Embodiment Synthesis (3R, 4R, 5S) -4-acetamido-5- (N-acetimidamide) -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester Hydrobromide (2)
1 g of oseltamivir (3.2 mmol) is dissolved in 10 ml of ethanol and the mixture is cooled to 0 ° C. 1.04 g of S- (naphthylmethyl) acetimidobromide (1.1 equiv) is added to the solution and then stirred at room temperature for 1 hour. Subsequently, the mixture is concentrated in vacuo and dissolved in about 80 ml of water. The solution is washed with a little diethyl ether and concentrated in vacuo. At this point, the product (85%) still contains a small amount of the parent compound, which can be removed only by column chromatography (DCM / MeOH, 5-10%). Yield: 960 mg (71%) (white solid).
DC: _Rf = 0.65 (DCM / MeOH, 9: 1)
¹ H-NMR (DMSO-d ₆ , 300 MHz):
δ / ppm = 0.79 (t, ³ J = 7.4 Hz, 3H), 0.85 (t, ³ J = 7.4 Hz, 3H), 1.23 (t, ³ J = 7.1 Hz, 3H) ), 1.44 (m _c , 4H), 1.83 (s, 3H), 2.11 (s, 3H), 2.33 (m _c , 1H), 2.67 (dd, ² J = 17 .6 Hz, ³ J = 4.7 Hz, 1H), 3.42 (quin, ³ J = 5.6 Hz, 1H), 3.82 (m _c , 1H), 4.05 (m _c , 1H), 4 .17 (q, ³ J = 7.1 Hz, 2H), 4.35, (m _c , 1H), 6.69 (m _c , 1H), 8.04 (br d, ³ J = 9.0 Hz, 1H), 8.63 (br s, 1H), 9.25, 9.35 (2xbr s, 1H).
MS (ESI): m / z = 354 [M + H] ⁺

(3R, 4R, 5S) -4-acetamido-5- (N-acetimidamide) -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid (3)
Oseltamivir amidine ethyl ester (217 mg, 0.5 mmol) dissolved in 10 ml MeOH was mixed with 1.5 ml 1 M methanol KOH (3 eq) and stirred at 40 ° C. for 1 h until starting material was no longer detected by DC. To do. The solution is diluted with water and the pH value is adjusted to 7-8 with 1M HCl. The solution is concentrated to dryness and the residue is then purified by reverse phase flash chromatography (RP-18 column, eluent: water, detection: iodine chamber). After lyophilization, the product is isolated as a white powder.
Yield: 88% (white fine powder).
¹ H-NMR (D ₂ O, 300 MHz):
δ / ppm = 0.84 (t, ³ J = 7.4 Hz, 3H), 0.89 (t, ³ J = 7.4 Hz), 3H, 1.38-1.63 (m, 4H), 2 .03 (s, 3H), 2.23 (s, 3H), 2.43 (m _c , 1H), 2.82 (dd, ² J = 17.5 Hz, ³ J = 4.8 Hz, 1H,) , 3.53 (quin, ³ J = 5.4 Hz, 1H), 3.93-4.09 (m, 2H), 4.36 m _c , 1H), 6.71 (brs, 1H).
MS (ESI): m / z = 348 [M + Na] ⁺ , 326 [M + H] ⁺ .
_{HRMS (ESI): C 16 H} 27 N 3 O 4 [M + H] + of m / z Calculated: 326.20743, found: 326.20737.

(3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidoamide] -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester ( 4)
465 mg oseltamivir (1.49 mmol), 290 mg DIPEA (389 μl, 1.5 eq) are dissolved in 5 ml dichloromethane and cooled to 0 ° C. Freshly prepared acetohydroxymoyl chloride (209 mg, 1.5 eq) is slowly added (dropwise) to this solution. The mixture is stirred at room temperature for 4 hours, mixed with 15 ml of water, stirred for a further hour and then separated on a separatory funnel. In order to increase the yield of the desired amidoxime, the aqueous phase is extracted four times with dichloromethane. The combined organic phases are dried over Na ₂ SO ₄ and concentrated in vacuo. The crude product is purified by column chromatography on silica gel (DCM / MeOH, 9: 1).
Yield: 70% (colorless crystalline solid)
DC: _Rf = 0.29 (DCM / MeOH, 9: 1)
¹ H-NMR (DMSO-d ₆ , 300 MHz):
δ / ppm = 0.80 (t, ³ J = 7.4 Hz, 3H), 0.85 (t, ³ J = 7.4 Hz, 3H), 1.23 (t, ³ J = 7.1 Hz, 3H) ), 1.43 (m _c , 4H), 1.80 (s, 3H), 1.95 (s, 3H), 2.38 (m _c , 1H), 2.62 (dd, ² J = 17 .4 Hz, ³ J = 5.0 Hz, 1 H), 3.40 (quin, ³ J = 5.6 Hz, 1 H), 3.65 (m _c , 1 H) 3.78 (dd, ² J = 17.4 Hz ^{, 3 J = 8.7Hz, 1H)} , 4.15 (q, 3 J = 7.1Hz, 2H), 4.19 (m c, 1H), 6.67, (m c, 1H), 6. 81 (brd, 1H, ³ J = 9.1 Hz), 7.99 (d, 1H, ³ J = 8.6 Hz), 9.73 (br s, 1H).
MS (ESI): m / z = 392 [M + Na] ⁺ , 370 [M + H] ⁺ , 354 [M-OH + H] ⁺ .
_{HRMS (ESI): C 18 H} 31 N 3 O 5 [M + H] + of m / z Calculated: 370.23365, found: 370.23379.

(3R, 4R, 5S) -4-acetamido-5- [N- (N′hydroxy) guanidino] -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester (6)
Dissolve 213 mg cyanamide (0.6 mmol) in 5 ml anhydrous dioxane and add exactly 1 equivalent of free hydroxylamine (20 mg). It is stirred for 30 minutes at room temperature, concentrated and a white solid is obtained after several additions and removals of dichloromethane and diethyl ether.
Yield: 222 mg (100%) (white solid)
DC: _Rf = 0.20 (EtOAc / MeOH, 6: 4)
¹ H-NMR (DMSO-d ₆ , 300 MHz):
δ / ppm = 0.80 (t, ³ J = 7.3 Hz, 3H), 0.84 (t, ³ J = 7.4 Hz, 3H), 1.24 (t, ³ J = 7.1 Hz, 3H) ), 1.40 (m, 4H), 1.83 (s, 3H), 1.99-2.07 (m, 1H), 2.86 (dd, ² J = 16.7 Hz, ³ J = 2 .5 Hz, 1 H), 3.38 (quin, ³ J = 5.5 Hz, 1 H), 3.49 (m, 1 H), 3.80 (m, 1 H), 4.01, (m, 1 H), 4.14 (q, ³ J = 7.1 Hz, 2H), 4.24 (m, 1H), 4.92 (s, 2H), 6.64 (m, 1H), 7.72 (br s, 1H), 7.79 (d, ³ J = 8.8 Hz, 1H).
MS (ESI):
m / z = 741 [2M + H] ⁺ , 393 [M + Na] ⁺ , 386, 371 [M + H] ⁺ .
HRMS (ESI): C ₁₇ H ₃₀ N ₄ O ₅ [M + H] ⁺ m / z calculated: 371.22890, found: 371.222911

(3R, 4R, 5S) -4-Acetamido-5- [N- (N′-n-hexyloxycarbonyl) thioureido] -3- (1-ethylpropoxy) -cyclohex-1-ene-1-carboxylate Ester (7)
500 mg of oseltamivir (1.6 mmol) is dissolved in 50 ml of anhydrous dichloromethane and an equimolar amount of hexyloxycarbonyl isothiocyanate (approximately 0.5 M in dichloromethane) is slowly added dropwise. After stirring for 2 hours at room temperature, it is washed with 1% HCl, water, NaCl solution. The organic phase is dehydrated with Na ₂ SO ₄ and concentrated on a rotary evaporator. The crude product can be finely ground or washed with cyclohexane, which makes it sufficiently pure for the next reaction. The compound was further purified by silica gel column chromatography (Cy / EtOAc, 6: 4) for elemental analysis.
Yield: 600 mg (75%) (white to yellowish solid)
DC: _Rf = 0.20 (Cy / EtOAc, 6: 4)
¹ H-NMR (DMSO-d6,300 MHz):
δ / ppm = 0.79 (t, ³ J = 7.4 Hz, 3H), 0.84 (t, ³ J = 7.3 Hz, 3H), 0.87 (t, ³ J = 6.8 Hz, 3H) ), 1.23 (t, ³ J = 7.2 Hz, 3H), 1.30 (m _c , 6H), 1.45 (m _c , 4H), 1.57 (m _c , 2H) 1.80 (S, 3H), 2.30 (dd, 1H, ² J = 17.8 Hz, ³ J = 6.8 Hz), 2.90 (dd, 1H, ² J = 17.8 Hz, ³ J = 5.0 Hz) ), 3, 43 (quin, 1H, ³ J = 5.4 Hz), 4.07 (m _c , 4H), 4.16 (q, 2H, ³ J = 7.1 Hz), 4.55 (m _c ^{, 1H), 6.74 (br s} , 1H), 7.91 (br d, 1H, 3 J = 8.0Hz), 9.98 (d, 1H, 3 J = 7.6Hz), 10.90 s, 1H).
MS (ESI):
m / z = 500 [M + H] ⁺ , 483, 412

(3R, 4R, 5S) -4-acetamido-5- [N- (N′-n-hexyloxycarbonyl)-(N ″-(2-methoxypropan-2-yl) oxy) guanidino] -3- ( 1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester (8)
358 mg oseltamivir hexylthiourea (0.72 mmol) was dissolved in 10 ml anhydrous dichloromethane, 151 mg O- (2-methoxypropan-2-yl) hydroxylamine (2 eq), 251 μl DIPEA (2 eq), 276 mg Of EDCI (2 eq) is added. The mixture is stirred at room temperature for 1.5 days, concentrated and treated by column chromatography on silica gel (DCM / MeOH, 0-2%).
DC: _Rf = 0.39 (DCM / MeOH, 98: 2)
Yield: 374 mg (91%) (white solid) (store this at -20 <0> C)
¹ H-NMR (DMSO-d ₆ , 300 MHz):
δ / ppm = 0.83 (m _c , 9H) 1.27 (m _c , 12H), 1.27 (m _c , 12H), 1.45 (m _c , 4H), 1.57 (m _c , 2H), 1.80 (s, 3H), 2.30 (dd, 1H, ² J = 18.1 Hz, ³ J = 7.1 Hz), 2.90 (dd, 1H, ² J = 18.1 Hz, ³ J = 5.2 Hz), 3.06 (s, 3H), 3.43 (quin, 1H, ³ J = 5.6 Hz), 4.06 (m _c , 4H), 4.16 (q, 2H) , ³ J = 7.1 Hz), 4.55 (m _c , 1H), 6.74 (s, 1H), 7.91 (d, 1H, ³ J = 8.1 Hz), 9.98 (d, 1H, ³ J = 7.8 Hz), 10.90 (s, 1H).
_{HRMS (ESI): C 28 H} 50 N 4 O 8 [M + H] + of m / z Calculated: 571.37014, found: 571.37034.

(3R, 4R, 5S) -4-acetamido-5- (N-guanidino) -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid (9)
A) Pharmacokinetic characterization of inhibitors and prodrugs Stability studies over 6 hours of amidine active form (3) and its prodrug (2, 4) and guanidine active form (9) and its prodrug (6, 10) 0.2 mM in 50 mM potassium phosphate buffer A stability test was performed at the concentration. For this purpose, a 2 mM stock solution was prepared with 10 mM potassium phosphate buffer (pH 7.4) and diluted 1:10 with phosphate buffer at each pH value. Each compound was tested at pH 2.0, 7.4 and 9.0. For this purpose, samples were analyzed every 30 minutes by HPLC and stability was tested for 6 hours. The concentration at t = 0 minutes was set as 100%.

  In addition, the substance was tested in human and mouse plasma. For this purpose, 630 μl of plasma was mixed with 70 μl of a 2 mM stock solution of each compound dissolved in 10 mM phosphate buffer (pH 7.4). Incubations were performed at 37 ° C. in a shaking water bath. At 15 min, 30 min, 45 min, 60 min, 90 min and 120 min time points, the incubation was terminated by removing 100 μl of sample and adding 100 μl of acetonitrile. Samples were centrifuged (12,000 rpm / 10 min) and the supernatant was examined by HPLC.

  The following HPLC method was used to analyze the stability study.

HPLC Method HPLC-Systems Waters Autosampler 717plus, Waters 600 Controller, Waters 600 Pump, Waters 2487 Dual λ Absorbance Detector and EZChrom Elite Client / Server Recording and Analysis Software (Version 2.8.3)
Column: LiChrosphere 60 RP-select B (125 × 4 mm, 5 μm) with RP-select B guard column (4 × 4 mm).
Flow rate: 1 ml / min Eluent: In the case of 3, 9, 60% 10 mM KH ₂ PO ₄ /0.1% TFA pH 3.0
40% MeOH,
In the case of 2, 4, 6, 10, 50% 10 mM KH ₂ PO ₄ /0.1% TFA pH 3.0
50% MeOH
Run time: 7.5 minutes Detection: 230 nm
Injection volume: 10 μl
Retention time: 4 4.2 ± 0.1 minutes
9 4.4 ± 0.2 minutes
3 4.5 ± 0.2 minutes
6 4.6 ± 0.1 min
2 4.8 ± 0.1 minutes
10 3.7 ± 0.1 min

2 weeks stability study of amidine active form (3) and guanidine active form (9)
Study at pH 7.4:
The storage stability of the dissolved form was measured at a concentration of 0.2 mg / ml. For this purpose, the compounds are dissolved in 50 mM KH ₂ PO ₄ buffer (pH 7.4) or aqua bidest and at room temperature (RT) (pH 7.4) over the study period or in the refrigerator at 4 ° C. (pH 7. 4 or aqua bidest). The concentration of the active form was measured by HPLC after 12 hours, 1 day, 2 days, 4 days, 7 days and 14 days.

2. Test compound solubility assay
Measurement of solubility at different pH values:
The solubility of the compounds was measured with phosphate buffer at different pH values (2.0, 7.4 and 9.0). For this purpose, several mg of compound was weighed and mixed with a fixed volume of 50 mM KH ₂ PO ₄ buffer at each pH value in a 50 mM solution. If the compound did not dissolve completely, the suspension was shaken for 30 minutes. Subsequently, the undissolved portion was removed by centrifugation at 10,000 rpm for 15 minutes, and the concentration of the supernatant was measured by HPLC.

3. Measurement of protein binding of amidine active form (3) and its prodrug (2, 4) and guanidine active form (9) and its prodrug (6, 10) at three different concentrations (10, 25, and 50 μM) Plasma protein binding was performed. A 4% albumin solution was used as the protein solution. In each case, 50 μl of a 10-fold concentrated substance solution was pipetted into 450 μl of protein solution. Incubation was for 15 minutes at 37 ° C. in a shaking water bath. Subsequently, the sample was transferred to an ultrafiltration unit (Vivaspin 500, 10 kDa cut-off) and centrifuged at 10,000 rpm for 15 minutes. The filtrate was analyzed by HPLC. In addition, a control was provided for each concentration (this was not treated with protein and not centrifuged). However, another control without protein supernatant was centrifuged in a filtration unit, indicating that this prodrug was not retained by the membrane and was used to validate this procedure.

In addition, the protein binding of the two active forms (oseltamivir-amidine (3) and oseltamivir-guanidine (9)) was examined in human plasma. For this purpose, human plasma was used instead of 4% albumin solution. Protein binding was determined to be 3.7 ± 1.4% for compound 3 and 8.6 ± 3.0% for compound 9. As expected, these values are somewhat higher than those obtained with the 4% albumin solution, due to the presence of other plasma proteins in addition to albumin (eg, α ₁ acidic glycoprotein).

4). Investigation of in vivo activation of various prodrugs (2, 4, 6, 10)
Measurement of prodrug activation by various intracellular enzyme systems:
Activation of prodrugs to their active form was measured in vitro by intracellular enzyme preparations. As enzyme preparations, 9000G supernatants, microsomes and mitochondria from human and porcine liver tissue were used. The incubation mixture consisted of 500 μM prodrug, 500 μM NADH, 1 U esterase and 0.3 mg enzyme preparation dissolved in 150 μl 100 mM phosphate buffer (pH 6.3). Incubation was for 30 minutes at 37 ° C. in a shaking water bath. Incubation was terminated by adding 150 μl of acetonitrile. The sample was then shaken for 10 minutes and centrifuged for 15 minutes at 10,000 rpm to remove the precipitated protein. The supernatant was measured by HPLC.

HPLC method HPLC system for determination of active form (3/9) and prodrug (2, 4/6, 10) Waters Alliance HPLC system with Waters e2695 XC separation module,
Waters 2998 photodiode array detector and Empower2 software column: LiChrosphere 60 RP-select B (125 × 4 mm, 5 μm) with C18 guard column (4 × 4 mm)
Flow rate: 1 ml / min Mobile phase: 70% 10 mM KH ₂ PO ₄ /0.1% TFA pH 6.5
30% MeOH
Run time: 12 minutes Detection: 210nm
Injection volume: 10 μl
Retention time: 3 4.8 ± 0.2 minutes
9 4.7 ± 0.2 minutes
10 24.8 ± 0.4 minutes
6 25.2 ± 0.3 minutes
4 26.6 ± 0.3 minutes
2 26.6 ± 0.3 minutes

Antiviral activity
Measurement of antiviral activity in a chemiluminescence-based neuraminidase (NA) -inhibition assay The following influenza viruses were used in the study:
H1N1 viruses: A / Jena / 5258/2009, A / Jena / 5555/09, A / HH / 1580/09, A / 342/2009 (oseltamivir resistance)
H2N3 viruses: Hong Kong / 8/68, Saxony / 6/02, Berlin / 10/04, Rhineland-Pfalz / 3911/03.

  A commercially available NA-Star kit (Tropix, Applied Biosystems, Darmstadt) was used to examine the inhibition of viral neuraminidase by test compounds 3 and 9 and the control substance.

  According to the manufacturer's recommendations, the optimal dilution of test virus for subsequent inhibition assays was first determined in a preliminary test. For this purpose, the virus suspension was diluted with NA-Star buffer (dilution factor 3) in the absence of neuraminidase inhibitor (NAI). Subsequently, NA dilution assays to determine 50% inhibitory concentrations used virus dilutions that resulted in a 40: 1 signal background ratio.

  For the NA inhibition assay of 6 virus controls / plate, 25 μl of assay buffer or 25 μl in assay buffer containing test substance (3 parallel / dilution) or control substance (2 parallel / dilution) was added to Applied to individual wells of the titer plate. 25 μl of virus dilution was then added to each well. After an incubation time of 20 minutes at 37 ° C., the substrate was diluted 1: 500 with assay buffer and 10 μl was added to each well respectively. After 30 minutes, chemiluminescence measurement was performed with a plate reader (microtiter plate luminometer, Dynax Technology). For assay evaluation, the average of chemiluminescence measured for 6 untreated virus controls was taken as a value of 100% of NA activity and used to calculate the relative NA activity of individual wells treated with the substance. Subsequently, 50% inhibitory concentrations (IC50) of the test substance and the control substance were calculated from the resulting average dose response curves of two independent assays by linear interpolation of EXCEL.

Measurement of antiviral activity of test substances in virus production (VY) inhibition assay
Cells: MDCK cells
Influenza virus: a) A / Jena / 5258/2009 (epidemic H1N1; oseltamivir sensitivity)
b) A / 342/2009 (H1N1; oseltamivir resistance)

  By adding an antiviral agent (100 μl / well; 3 parallel / concentration / test substance and 2 parallel / concentration / control substance, dilution factor 10), viral replication can be selectively inhibited. This can be measured experimentally based on a decrease in virus titer in the supernatant.

  In this assay, a two-day-old closed cell monolayer was inoculated with a fixed dose of virus, and the three untreated virus controls resulted in an incomplete cytopathic effect 48 hours after infection. After 1 hour incubation at 37 ° C., each well was washed 3 times in succession to remove virus that did not bind to the cells and 100 μl of test medium (cell and virus control) or dilutions of the substance were added. Added. After incubation at 37 ° C. for 48 hours, the supernatant of each well was collected for measuring the virus titer of the supernatant.

Viral titer measurements were performed on 2-day old MDCK cell monolayers in microtiter plates. For this purpose, log serial dilutions (maximum dilution factor 10; maximum dilution 10 ⁻⁷ ) were first made from the supernatant from the VY inhibition assay. These were seeded into cells (4 wells / virus dilution each) and incubated at 37 ° C. for 4 days. During this period, a cytopathic effect was formed. The cells were fixed and stained with a crystal violet formalin solution, and then visually evaluated on a light box.

  Subsequently, virus titers were calculated according to Reed and Muench. To calculate the titer reduction, the average virus titer of the three virus controls was taken as 100%.

Measurement of the antiviral activity of test substances in the cpE inhibition assay The replication of the virus used in the assay leads to the complete destruction of the host cells due to a very pronounced cytopathic effect (cpE). By adding an antiviral active substance (100 μl / well; 3 parallel / concentration, dilution factor 2), virus-induced cpE can be selectively inhibited. In the assay, untreated and substance-treated closed cell layers were inoculated with a fixed dose of virus, while the untreated virus control resulted in complete cpE 48 hours post infection. At this point, the remaining adherent cells were fixed and stained with a crystal violet / formalin solution. After elution of the dye, the inhibition of virus-induced cpE was measured photometrically with a Dynatech plate reader.

  Antiviral effect calculations were performed by comparing the optical density of the material-treated and untreated virus-infected cells with the average optical density of the cell control (set as 100%). Based on the average dose response curve of two experiments, the dilution (IC50) that inhibited the formation of virus-induced cpE by 50% was calculated by linear interpolation of EXCEL.

Cytotoxicity assay to determine 50% cytotoxic dose (CC ₅₀ ) of test substance in MDCK (Maidin Derby canine kidney) cell layer MDCK cells were seeded in microtiter plates, 5% CO ₂ , 37 ° C. and Incubation was carried out for 48 hours in a 95% humidity incubator to form a closed cell layer. For this purpose, the medium was removed and the substance was applied to the culture medium at various concentrations (100 μl / well, 3 parallel / concentration, dilution factor 2). For the determination of control values (6 untreated cell controls), 100 μl of medium was used each. The cells are stained with crystal violet / methanol 72 hours after incubation with administration of the substance. After elution of the dye, the optical density (OD) of each well was measured with a Dynatech plate photometer (550/630 nm) and compared to the cell control average. The average of the controls was 100%.

Animal research
Animal Handling / Preparation Sprague Dawley (SD) rats weighing approximately 300-350 g were acclimated for 10 days prior to the start of the experiment and placed in an air-conditioned room at a constant temperature (20 ° C.) and a humidity of 50%. In this room, the light / dark cycle was 12 hours. The dark period started at 18:00 every day and switched to the light period at 6:00. During the acclimation period, rats are placed in a standard size 3 cage (length: 42 cm, width 26 cm, height: 15 cm) and transferred to a special laboratory under the same environmental conditions 3 days before the start of the experiment. did. Rats were given a free maintenance diet (maintenance diet for rats and mice; No. 1320; Altromin) and tap water.

  The animal experiments described herein were conducted in accordance with the “NIH Guidelines” and corresponding regulations for the handling and use of laboratory animals after approval by the Ministry of Agricultural, Environmental and Rural Area of Schleswig-Holstein.

  Catheters were implanted in the femoral vein and femoral artery of rats that received intravenous administration (iv). Rats that received only oral administration of the substance received only an intravenous catheter.

  Rats were anesthetized with pentobarbital (60 mg / kg intraperitoneal (ip)) and further anesthetized with diethyl ether when the depth of anesthesia was insufficient. After the neck and the right inguinal region were shaved, the rat was placed in a supine position and placed on an electric heating stage (EBERLE, type 52102) to maintain body temperature, and the hind limbs were fixed. An incision about 1.5 cm long was made along the groin. Subsequently, the femoral artery, femoral vein, and femoral nerve vessel bundle were incised to a length of about 1 cm.

  After separation of the femoral vein, a cotton thread was placed near its periphery and reversibly occluded by tightly tying the blood vessels. A blood vessel approximately 5 mm in the distal direction was tied with a second thread to create congestion. Using vascular scissors, make a small incision (about 1/3 of the total length of congestion from terminal ligation) to a blood vessel in the congested region, and a polyethylene tube (length: 26 cm; ID: 26 cm; ID : 0.58 mm, OD 0.96 mm) was inserted 3 cm near the vena cava using a container dilator. The catheter was secured to the vessel with proximal and distal threads.

  In contrast to the femoral vein, the femoral artery was first occluded by terminal ligation and then confined nearby by tightly tying the thread. Again, the catheter was implanted as described above. Since the inner diameter of the artery is small, a specially made of a polyethylene tube (length: 26 cm, ID: 0.58 mm, OD 0.96 mm) and a welded polyethylene tube of 3 cm length (ID: 0.28 mm, OD: 0.61 mm) An arterial catheter was used.

  After incorporating the catheter, the animal was placed prone and a 5 mm wide incision was made in the neck. Using a metal rod and tube, the catheter sealed with a wire pin was withdrawn from the projection of the neck, and the neck was fixed with cotton thread and cut to a length of about 3 cm.

  Again in the supine position, the subcutaneous fat and then the epidermis were first stitched together with 3-4 double buttonhole sutures and disinfected with Betaisadona® solution. The next day, the catheters were each washed in the morning and evening with 300 μl of heparin solution (250 IU / ml). The operated rats were individually placed in a Plexiglas experimental cage with dimensions of height: 20 cm, width: 22 cm, and length: 25 cm, or a standard size 3 cage from the day of catheter insertion.

  The animals with the inserted catheters were placed in the laboratory for 1 day after surgery and individually placed in their experimental cages. The test compound was applied on the second day after surgery. On the day of the experiment, the rats were weighed 1 hour before the test and the arterial catheter was washed with 300 μl of heparin solution. Subsequently, the compound was administered intravenously (iv) or orally.

Implementation of animal studies Oseltamivir derivative 3 was administered intravenously to 5 rats at a concentration of 10 mg / kg. Neuraminidase inhibitor (2, 4) was orally administered to 5 or 6 rats at a dose of 50 mg / kg. In addition, derivative 3 was orally administered (50 mg / kg) to 3 rats. Oral administration was performed by gavage as a suspension or solution containing gum arabic (10% w / v).

  Oseltamivir derivative 9 was administered intravenously to 5 rats at a concentration of 10 mg / kg. Neuraminidase inhibitor (6, 10) was orally administered to 4 or 5 rats at a dose of 50 mg / kg. In addition, derivative 9 was orally administered (50 mg / kg) to 3 rats. Oral administration was performed by gavage as a suspension or solution containing gum arabic (10% w / v).

  Plasma samples were collected at 5 minutes, 10 minutes, 20 minutes, 45 minutes, 90 minutes, 150 minutes, 240 minutes and 360 minutes after intravenous (iv) administration, 30 minutes after oral administration, 60 minutes Samples were taken at minutes, 90 minutes, 120 minutes, 180 minutes, 240 minutes and 360 minutes. For this purpose, in each case 300 μl of whole blood was collected using an insulin syringe and transferred to EDTA-coated Microvettes CB 300 (Sarstedt, Numbrett). After each harvest it was washed with 100 μl 0.9% saline or heparin solution (250 IU / ml) every 60 minutes. The blood sample was briefly shaken and placed on ice until centrifugation (4 ° C .; 14000 U / min; 10 minutes). Subsequently, the sample was frozen at −80 ° C.

  Six hours after drug administration, sacrifice was performed by decapitation with guillotine. Subsequently, the organ was removed. All organs were disinfected and frozen with 2-methylbutane cooled with dry ice. Liver, kidney and lung were collected.

Analysis of plasma samples Plasma samples were processed and analyzed by HPLC. For this purpose, plasma samples were thawed at room temperature. In each case, 80 μl of methanol (+ 0.2% TFA) was prepared, followed by pipetting 80 μl of plasma sample. Samples were shaken for 45 minutes to precipitate plasma proteins. Samples were frozen at −80 ° C., thawed and shaken for an additional 15 minutes. The sample was centrifuged at 13,000 RPM for 15 minutes and the supernatant was transferred to an HPLC vial. In each case, 50 μl was used for measurement by LC / MS.

Animal studies were evaluated using the following LC / MS method.
LC / MS HPLC system: Agilent 1100 binary pump, Agilent 1100 diode array detector, Agilent 1100 well plate autosampler, degasser G1322A
Column: LiChrosphere 60 RP-select B (125 × 3 mm, 5 μl) with RP-select; B guard column (4 × 4 mm)
Mass spectrometer Esquire-LC
Interface: ESI (Electron Impact Ionization)
Nebulizer: 0.28 MPa (40.0 psi)
Dry gas: 8.0 ml / min Drying temperature: 350 ° C.
HV capillary 5000V
Mobile phase: A 0.1% TFA aqua bidest solution (pH 2.5)
B 0.1% TFA MeOH solution gradient profile: Time A [%] B [%]
0 55 45
8 25 75
10 25 75
11 55 45
17 55 45
Flow rate: 0.3 ml / min Run time: 17 minutes Detection: PDA (190-400 nm)
Injection volume: 50 μl
Retention time:
3 5.1 ± 0.3 minutes
9 5.1 ± 0.3 minutes
11 5.2 ± 0.3 minutes
12 5.2 ± 0.3 minutes

References
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8.Shie, JJ; Fang, JM; Wang, SY; Tsai, KC; Cheng, YS; Yang, AS; Hsiao, SC; Su, CY; Wong, CH Synthesis of tamiflu and its phosphonate congeners possessing potent anti-influenza activity J Am Chem Soc 2007, 129, 11892-3.
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Claims (9)

Amidoxime (3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidamide] -3- (1-ethylpropoxy) cyclohex-1-ene represented by the following structural formula -1-carboxylic acid ethyl ester,

Or (3R, 4R, 5S) -4-acetamido-5- (N-acetimidamide) -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid represented by the following structural formula,

And / or a pharmaceutically acceptable salt, solvate, and / or R / S enantiomer thereof.   2. A compound according to claim 1 for use as a medicament. General structural formula

(Wherein R ¹ is H;
R ² is H or

CH ₂ OH, CH ₂ NH ₂ , CH ₂ CONH ₂ , CH ₂ CH ₂ CONH ₂ , consisting of branched or unbranched, saturated or unsaturated, substituted or unsubstituted hydrocarbon chain having a chain length of 1-12 A substituent selected from the group;
R ³ is H, OH or O C (Me) ₂ OMe,
R ⁴ is H or CH ₂ CH ₃
R ⁵ is CH ₃
R ⁶ is CH ₃
X is

And
Z represents C, S, SO, P, PO), an influenza neuraminidase inhibitor for treating influenza infections, and pharmaceutically acceptable salts, solvates, or R / S enantiomers thereof. Amidoxime (3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidamide] -3- (1-ethylpropoxy) cyclohex-1-ene represented by the following structural formula -1-carboxylic acid ethyl ester,

Or (3R, 4R, 5S) -4-acetamido-5- (N-acetimidamide) -3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid represented by the following structural formula,

The influenza neuraminidase inhibitor for treating influenza infection according to claim 3, which is a pharmaceutically acceptable salt or solvate thereof.   Amidoxime (3R, 4R, 5S) -4-acetamido-5- [N- (N′-hydroxy) acetimidamide] -3- for preparing a pharmaceutical composition for use in the treatment of influenza infections (1-Ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester, (3R, 4R, 5S) -4-acetamido-5- (N-acetimidamide) -3- (1-ethylpropoxy) Cyclohex-1-ene-1-carboxylic acid, hydroxyguanidine (3R, 4R, 5S) -4-acetamido-5- [N- (N′hydroxy) guanidino] -3- (1-ethylpropoxy) cyclohex-1- Ene-1-carboxylic acid ethyl ester or (3R, 4R, 5S) -4-a Toamido-5-(N-guanidino) -3- (1-ethyl propoxy) cyclohex-1-ene-1-carboxylic acid, and their pharmaceutically acceptable salts or the use of solvate. General structural formula

A method for preparing a compound according to claim 1 or an inhibitor according to claim 3 or 4 comprising reacting oseltamivir of the following steps:
Reacting at least oseltamivir with acylhydroxymoyl chloride in an organic solvent at room temperature.   The method of claim 6, wherein the organic solvent is dichloromethane. The method according to claim 6 or 7, wherein the inhibitor is an amidoxime according to claim 3 (R ³ = OH). 3. Reaction of oseltamivir (3R, 4R, 5S) -4-acetamido-5-amino-3- (1-ethylpropoxy) cyclohex-1-ene-1-carboxylic acid ethyl ester A process for the preparation of a compound according to claim 3 or an inhibitor according to claim 3 or 4, comprising the following steps:
i) reacting oseltamivir with cyanobromide in an organic solvent at room temperature ii) reacting cyanamide formed with i with hydroxylamine in dioxane at room temperature.

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